Mushroom Biotechnology: Developing Cultivation Protocol for Four Different Mushrooms and Accessing Their Potential in Pollution Management

Abstract

The proteinaceous food value of mushrooms is well recognized and it may offer effective and lasting solutions to the problems of child malnutrition and protein supplement in pregnant ladies. Advancements have been made toward understanding mushroom biology, cultivation aspects, using a variety of agro lingo-cellulosic waste, breeding high yielding varieties, medicinal implications and uses of these unique fruiting bodies in bioremediation and waste water management. Mushrooms contain antioxidants and anticancerous chemicals in significant quantities. Use of Chaga mushroom in corona virus disease control has been suggested recently in Russia. After first cultivation of rat ear fungus (Auricularia auricula) in 600 A.D., now more than 20 species are commercially cultivated and protocols to culture about 300 mushrooms is now known. New cultivation methods are developed and discussed for Shiitake mushroom (Lentinula edodes), Lenzites sterioides, Reishi mushroom (Ganoderma lucidum) and Turkey tail (Trametes versicolor).

The biological efficiency was 45% for Lentinus and 56% for Reishi in experiments conducted at Botany Department of the M.S. University of Baroda. An increase in yield was recorded when Lentinus mycelium was exposed to blue light and 5–10 ppm Veradix (IBA). The cultivation of medicinal mushroom is profitable as well fascinating since it requires a range of specific environmental conditions such as humidity, temperature, etc. The efforts have been made to eliminate the use of polythene bags by using earthen pots in case of oyster mushroom. A range of substrates have been used to increase the yield and manage agro-waste produced in large quantity in different countries. Cellulose and hemicellulose served as better sources of mushroom production, whereas, in lignin containing substrates the growth was slower. Apart from using substrates, dilute acid soaking of the leaves produced better growth of oyster. Light and temperature levels are also critical for example Lentinus cultivation required 15–20 °C temp, while it was 20–25 °C in certain other species. For G. lucidum the temperature required was 30 °C. Exposure to light acts as a shock to switch over the mycelium from vegetative to reproductive stage. The efforts are being made to increase number of fruiting bodies by the use of Ni and Sn salts. This chapter deals with spawn production and the advances in cultivation of four medicinal mushrooms made in different parts of the world. Mushrooms breeding and strain improvement has resulted into many new and high yielding strains. Production of oyster mushroom is done in almost all parts of the country, extensive fungal surveys are needed for developing better conservation strategies.

1 Introduction

Fungi are marvelous and unique creation of nature to sustain it for long. Mushrooms are highly evolved fungi with fruiting bodies. These can be described as, “macrofungi with a distinctive fruiting body, large enough to be seen with naked eye and to be picked by hand” (Chang and Miles 1992). Mushrooms have been recognized by Food and Agriculture Organization as Food , contributing to ameliorate the protein malnutrition in countries which are largely cereal dependent. It is said that during Roman times one of the Caesars described mushroom as, “The foods of the Gods,” and promptly reserved them for himself, his ministers and soldiers. Breeding of edible mushrooms with the latest research methods and technologies will undoubtedly act as a boost for breeders to enhance upon the quality traits of the natural trash burners.

In many developing countries of Africa and Asia, large population still lives in tribal or rural areas. In Africa, more than seven in ten poor people live in rural regions and are dependent on small-scale agriculture (IFAD 2001). In the past decade, over 350 million people have been lifted out of poverty, but global poverty remains a massive and predominantly rural phenomenon, with 70% of the developing world’s 1.4 billion extremely poor people living in rural areas. The IFAD report (International Fund for Agricultural Development (IFAD) 2011) also describes challenges, including the high poverty rate in Sub-Saharan Africa and the lack of progress in South Asia. The report underscores threats to rural development posed by climate change, volatile food prices, and natural resource constraints (International Fund for Agricultural Development (IFAD) 2011). If the natural resources base is not managed for the long term, and is exploited for short-term gains, it will never help in economic development on the scale demanded to relieve poverty over past 50 years. The ecosystems have managed to supply food , fuel fodder and fiber and other ecosystem services across the world. The use of fruits, vegetables and mushrooms can aid in providing balanced diet to millions of under-nourished people. Mushrooms were earlier classified as plants, but since they do not use light energy and no photosynthesis occurs, they were reclassified as Fungi. However, they can produce several groups of enzyme complexes, which can convert the huge lignocellulosic waste materials into a wide diversity of products. These products have multibeneficial effects for human welfare (e.g., as food , tonic, medicine, feed and fertilizers for plant growth, and at the same time help in protecting and regenerating the environment). Total 148,000 species of fungi have been named to date, of an estimated 2.2–3.8 million. Fungi that might prove useful for bioenergy are therefore more likely to be discovered by broad, high throughput genomic screening programs. Fungi have great potential within the bioenergy sector. A mere 285 of 148,000 described fungal species are assessed on the Red List, equating to 0.2% (Antonelli et al. 2020). Moreover, cultivation and development of edible and medicinal mushrooms can positively generate equitable economic growth that has already had an impact at national and regional levels in this century. Along with ecofriendly agriculture there is a need for sustainable research and development of mushroom production (mushrooms themselves) and mushroom products (mushroom derivatives), which can become a “nongreen revolution” (Chang 1999, 2007).

In ancient times, mushrooms have been thought to have special powers. Their use in traditional ancient therapies dates back at least to the Neolithic age (Gargano et al. 2017). The Pharaohs of Egypt prized mushroom as a delicacy. The Egyptians thought mushrooms gave immortality to Pharaohs. It is mentioned in Ramayana that, when Lakshman was injured, Hanuman brought whole Kishkindha Mountain, he was told to search the medicine probably in luminous fungi/mushroom. Vedas tell us about “Soma drink” which may be Amanita or Psilocybe like hallucinogenic mushroom. Contemporary research has validated and documented much of the ancient knowledge on medicinal mushrooms (MM).

In Colombia by the year 2004 about 27 different species of Psilocybe type mushrooms were reported (Martinez 2020). In Manizales, Colombia, Psilocybe zapotecorum had been collected. Research on the subject has advanced further and the latest discoveries talk about mushroom cults that were held in countries such as Siberia, Greece, Japan and the exotic islands of Hawaii. The Amanita muscaria cult traced and detected by Robert Gordon Wasson on in the Ojibway tribe in North America is remarkable (Wasson 1980). I had probed the Muisca mushroom cult by a complete study done by with the aid of Carl Ruck and Clark Heinrich. New evidence and study of pieces of the Museo del Oro in Bogota suggest that the mushroom cults were not only practiced by the Muiscas, but also by the Tayronas and the Tolimas. My idea of an Amanita Mushroom Cult in the Muiscas would not have been developed without the encouragement of this wonderful human being, researcher, and tomato lover. The tribes in question which I suggest practiced a mushroom cult are the Tayronas, the Muiscas and the Panches or Tolimas. I will call the last ones Tolimas for the sake of confusion. There is evidence from seeing the pieces of goldsmithing belonging to this particular tribes in the Museo del Oro, being the Tolima ones being the most powerful and less least stylized of all, while the Tayronas show the most artistic progress to the top (Martinez 2020).

Ganoderma strains used in oriental folk medicine refer to Reishi (Moncalvo 2000) (Fig. 16.8a). Mushrooms are rich in protein and are classified among vegetables. Out of 14,000 known mushrooms, 3000 are edible. About 100 mushrooms are commercially cultivated, and only ten species are used at industrial scale. Truffles (Tuber of Ascomycota) are hypogean, most delicious used in gourmet delicacies and are one of the costliest natural products. Being mycorrhizal they grow in close proximity of specific tree roots and are rarely cultivated artificially. More than 700 mushrooms are used in traditional system as medicine. Morchella or Guchhi is India’s most delicious mushroom. Medicinal mushrooms (MM) are comparable to “medicinal plants” and can be defined as macroscopic fungi, used for alleviation of diseases or in balancing a healthy diet. According to definition of herbal drugs dried fruit bodies, mycelia or spores are considered mushroom or fungal drugs (Gargano et al. 2017). MM are used in a variety of ways:

• (a) As dietary food

• (b) Dietary supplement

• (c) Mushroom pharmaceuticals

• (d) Natural biocontrol agents for plants

• (e) Cosmeceuticals

Scientific names of certain mushrooms and their uses are listed in Table 16.1 and Figs. 16.1 and 16.2.

Table 16.1 Common mushrooms, their scientific names, occurrence/association, and uses

Fig. 16.1

Hallucinogenic mushroom Psilocybe. (Source: S.N. Tyagi, Meerut, India)

Fig. 16.2

Uses of Pleurotus or Oyster mushroom

2 Wild Edible Mushrooms

In nature besides providing food for man and other animals these eukaryotic microbes play an important role in recycling the carbon, nitrogen and other elements through enzymatic breakdown of lignocellulosic plant waste and animal dung, which serve as substrate for these saprophytic fungi (Chang 1993). Fungi digest the complex substances and utilize these through absorptive nutrition for their growth. Owing to this function of mushrooms, Chang and Miles (1992) advocated the use of mushroom biotechnology to utilize agro-wastes like cereal straw, coffee, and coconut waste. Nisha (2011) made use of waste paper and cotton from offices and labs to produce Pleurotus . Further the spent mass can be utilized to produce cattle/poultry feed or utilized as compost to enhance crop yield. Mushrooms can be present in three ecological habitats (a) saprobic (b) ectomycorrhizal or (c) parasitic as in case of Ganoderma and Heterobasidion . The fruit bodies may be produced above or below ground (hypogean). Archeological record reveals that edible species were associated with people living 13,000 years ago in Chile, but it was in China where consumption of wild fungi was first reliably noted (Boa 2004) (Fig. 16.3).

Fig. 16.3

Medicinal uses of Ganoderma

Saini et al. (2018) has reported 126 taxa of Agaricus from India. The fruiting body of Agaricus consists of 85–87% water. It is rich in protein (40–45%), 3–4% carbohydrates, 6–8% dietary fiber, 3–4% lipids, and vitamins. Specially vitamin B1, B2, niacin, ergosterol, and linoleic acid (70–80%), aromatic compound and soluble sugars, arabinose, glucose, and trehalose (Bellini et al. 2003). Potassium is the main mineral in A. brasiliensis (Oliveira et al. 2010). Large amount of alanine (11.9%) and tyrosine (2.4%) was present in protein (Kawagishi et al. 1989). Supplementing the substrate with controlled liberation of urea and Mn (II)Cl shortens the crop period from 35 to 28 days for Pleurotus spp. and also increases mushroom productivity (Lelley and Janben 1993). Mushrooms have low energy levels hence beneficial for weight reduction, low purine helps to prevent gout and rheumatism, low sodium which benefits persons having high blood pressure. Moreover they have a unique flavor and few are delicious. Mushrooms are medicinal foods with eight important amino acids and have higher nutritional values than fish or beef (Chang and Bushnell 1996; Fekadu 2015) (Fig. 16.4).

Fig. 16.4

Habitat and examples of different mushrooms

Thick leafy vegetation, senescenced leaves and fallen woody material on the forest floor result in prolific growth of saprophytic fungi aided by substrate availability from microbial decomposition of organic matter. Fast growth of temperate tree species such as Pinus and Eucalyptus is possible due to the presence of ectomycorrhizae, which help the roots to access the nutrients immobilized within soil aggregates, otherwise inaccessible even to fine roots. Therefore a large number of ectomycorrhizal fungi and consequently mushrooms occur in temperate forests. Fungi help in seedling establishment. Collection have been made from Sweden, Munich (Germany) and North America. The species collected were Morchella spp. Cantharellus, Tricholoma spp. Boletus edulis and the collection for 1992 season had been 2 million kg at a value of $41.1 million (Kaul 2002). Total world production of mushrooms was 34 billion kg in 2013 (Royse et al. 2017).

In India—Agaricus campestris , Cantharellus spp., Coprinus spp., Lantinus subnudus , Termitomyces spp., Tricholoma, Boletus spp. and Tuber spp. are some common mushrooms found in India. In Kashmir out of 175 species of agaricoid and clavarivid fungi 77 were mycorrhizal and were also found elsewhere in India. The gamut of Agaricoid species present in India has been negatively influenced by the replacement of natural forest by exotic trees like conifers, Eucalyptus, Casuarina in the hills of South India. Species of Morchella were collected from nature in Kashmir and Himachal Pradesh and sold in market. Survey conducted in Panchmahal, Narmada, Dahod and Vadodara districts showed the presence of Auricularia , Aurificaria, four species of Ganoderma , two species of Lenzites, 12 species of Phellinus and Schizophyllum commune from different forest trees in Gujarat.

2.1 Mushroom: The Magic Store of Health Benefits

Total commercial mushroom production worldwide has increased more than 21 times in 35 years. From 350,000 tons in 1965 to 7.5 million tons in 2000 (Boa 2004). Edible, medicinal, and wild mushrooms are the three major components of the global mushroom industry. Combined, the mushroom industry was valued at approximately $63 billion in 2013. Cultivated, edible mushrooms are the leading component (54%) accounting for approximately $34 billion, while medicinal mushrooms make up 38% or $24 billion and wild mushroom account for $5 billion or 8% of the total (Royse et al. 2017). According to an old Chinese saying, “medicines and food have a common origin.” Mushroom products are used for food and medicinal purposes (Wasser 2002; Stamets 2000). The term mushroom nutraceuticals has been coined to embody both nutritional and medicinal features. Asian cultures have also used Reishi, rendered in jade, as a talisman worn around the neck. Sometimes, whole dried Reishi are placed in the home to ward off the evil. It may be used to prepare Herbal Meditation tea.

The antler-shaped mushroom is the most rare and valuable form of Reishi, known in China for over 4000 years as Ling-Zhi, herbalist called Reishi the herb of spiritual potency and used it promote good health and longevity in Japan. HIV /Aids and anti-retrieval drugs derived from mushrooms have been used in Africa. Bioactive compounds of Ganoderma and Lentinus are useful for HIV patients (Bisen et al. 2010). When Ganoderma capsules were given to HIV patients hemoglobin level increased from 0.5 to 1.1 mg. Reishi is used to treat liver disorders, hypertension, arthritis, and other ailments. Reishi Gano is a king of mushroom essence (G. lucidum ) widely known as King of herbs. It is effective in:

• 1. scanning diseases

• 2. cleaning toxin

• 3. regulating body functions

• 4. recovery of health

• 5. preserving youthfulness

Members of two Aphyllophorales were collected from the branches of Tamarindus indica and Alangium salvifolium, fungi Trametes gibbosa (Pers.) Fr. and T. lactinea (Berk.) Pat. were found respectively attached to these trees in the Ratanmahal wildlife sanctuary, in Panchmahal district of Gujarat (Arya et al. 2008). Used as MM the fruit bodies of T. gibbosa have the ability to confer a protective effect to blood vessels of rat in the carrageenan assay, suggesting their possible use in pathological disease conditions leading to endothelial damage (Czarnecki and Grzybek 1995). Polysaccharides extracted from the mycelial culture of T. gibbosa , when administered intraperitoneally into white mice at a dosage of 300 mg/kg inhibited the growth of Sarcoma 180 and Ehrlich solid cancers by 80% and 90%, respectively (Ohtsuka et al. 1973). The petroleum ether and ethyl acetate extract of T. gibbosa were shown to be cytotoxic to human cervix epitheloid carcinoma cell lines (Hela) and human hepatoma cell lines (SMMC-7721) using the MTT-dye assay. The methanolic extract, however, showed weak activity when compared with the flavonoid quercetin (Ren et al. 2006), and was inhibitory (i.e., <40% inhibition) on HIV-1 reverse transcriptase activity in vitro (Mlinarič et al. 2005). It was shown that organic extracts of T. gibbosa mycelium was effective in inhibiting the growth of K562 cells, a laboratory model of human chronic myelogenous leukemia (Yassin et al. 2008).

Ch. Ramesh and Pattar (2010) while study of antioxidant activity of six mushrooms found that the MIC values of the selected microorganisms on extract of Clavaria vermiculris and Marasmius oreades inhibited the growth of Gram-negative bacteria better than Gram-positive bacteria and yeast. According to Ishikawa et al. (2001) the antibacterial activities of aqueous extract of both edible Pleurotus sajor caju and Agaricus were significant up to 10% dilution against all the tested pathogens. Fungus Ganoderma showed antitumor activity (Kimura et al. 2002) hepatoprotective (Zhang et al. 2002), and high activity (62.5%), against bacteria followed by extracts from species of Rigidoporus (27.2%). A low percentage of antimicrobial activity was shown by extracts of Hymenochetous fungi Phellinus species (7.8%) (Tambekar et al. 2006) summarized the similarity of minimum inhibitory concentrations between the mushrooms extracts and positive control in relation to their susceptibility to the tested microorganisms. These results revealed that Lycoperdon perlatum , Clavaria vermiculris , Marasmius oreades , Pleurotus pulmonarius had comparatively similar concentrations of standard antimicrobials, which confirms the presence of bioactive components in edible mushrooms. The Staphylococcus aureus is widely recognized as an important food-borne pathogen, and the potential of its inhibition can be caused by Lentinus edodes may receive more attention (Getha et al. 2009).

2.2 Chemicals Present in Medicinal Mushrooms and Their Biological Activities

Mushrooms have medicinal value, which is generally attributed to the biochemicals present in them. Pleurotus is rich in fiber, chitin and beta β glucans, they contain polysaccharide —Protein Complex (PSPC) and lectins that have antitumor activity. Four mushrooms Lentinus, Schizophyllum, Grifola and Sclerotinia sclotiorum produce β glucans called lentinan, schizophyllan (also called SPG, sonifilan, or sizofiran), grifolan, and SSG are known for antitumor activity. Most of the β-(1-6)-branched β-(1-3) linked glucans are able to act as antitumor agents (Fekadu 2014). Along with lentinan and schizophyllan, antitumor glucans from G. frondosa and Trametes versicolor have been used in different clinical trials and led to an increase of survival rate and time in patients of different types of cancer, when used in combination with chemotherapy and/or surgery (Lindequist et al. 2005).

Reishi is rich in active organic compounds such as polysaccharides, amino acids, proteins, triterpenes, ascorbic acid, sterols, lipids, alkaloids, a glucose, a coumarine glycoside, volatile oil, riboflavin, and more. These compounds are being studied for their effects on the immune system and antitumor effect. Ethyl acetate, methanol, and aqueous extract of G. lucidum can highly inhibit O₂ and OH radicals, but aqueous extract cannot inhibit ferrous ion induced lipid peroxidation whereas ethanol extracts of the mycelium of G. lucidum has high antiperoxidative activity (Lakshmi et al. 2003). Ganoderan, the biopolymer produced by G. lucidum , has also been used as adjuvant therapy in combination with anticancer drugs. It was able to prolong the survival of Lewis carcinoma bearing mice and to increase the effectiveness of cytotoxic drugs and immunomodulatory in patient with prostate cancer (Vannucci et al. 2013). Polysaccharides present in G. lucidum have also shown prophylactic activity toward chemically injured macrophages (You and Lin 2002).

3 Molecular Characterization and Genetic Improvement

There has been a significant progress in genome analysis of cultivated mushrooms. Delignification of wood caused by most of the white-rot fungi occur due to laccases produced by bacteria, fungi, insects, and plants (Thurston 1994). Laccase production in different fungi occurs by more than on isozyme (Baldrian 2006; Karen et al. 2016). A number of genes encoding enzymes are involved in substrate utilization such as laccase, xylanase, and cellobiohydrolase. The transposons namely Abr1, abr3 and Tab1 have been identified and characterized in Agaricus bisporus (Yadav et al. 2007). Mushroom A. bisporus has haploid genome of 13 chromosomes, for Pleurotus ostreatus n = 11 and Volvariella volvacea it is 15 (Chiu and Moore 1999). Among the important genes include MAT—gene determining mating type, BSN—gene determining basidial spore number and cap color-determining locus PPC1. MAT is located on chromosome 1. The cap color is the most important phenotypic trait to distinguish wild and cultivated strains of A. bisporus . Amplified fragment length polymorphism (AFLP) markers were used for precise assessment of genetic variation in germplasm strains and newly developed hybrids, and identification of sexually incompatible strains in A. bitorquis was proposed by Yadav et al. (2006).

The potential of 5′ end of IGS2 region to identify the two mating types of Pleurotus pulmonarius was demonstrated by various analyses and examinations. The high variation among the sequences of IGS had previously been reported by Zhang et al. (2006), Guidot et al. (1999), Iracabal and Labarere (1994), and James et al. (2001). The amplification of 5′ end of IGS2 region could clearly identify two types of monokaryons (A & B) and was able to separate the dikaryotic cultures from monokaryotic which were wrongly isolated as monokaryon. Moreover, the pedigree tracking of a successfully crossed strain of A4 × B3 from two compatible mating partners has been demonstrated. Similar methodology had been employed by James et al. (2001) to discriminate the mono- and dikaryotic strains of the widespread mushroom Schizophyllum commune . In another study, Kavousi et al. (2008) could demonstrate that the homo- and heterokaryotic isolates are distinguishable by RAPD markers. According to Bhatt et al. (2018) members of Polyporaceae are polyphyletic, Trametes may be enlarged to include Lenzites.

In homothallism, the basidiospore germinates and form fruit body without the requirement of a mating process, so the whole life cycle can be completed with a single basidiospore. Based upon the number of basidiospore formation on a basidium and the involvement of incompatibility factor(s), the homothallism can be divided into two forms: primary and secondary. The phenomenon of primary homothallism in Volvariella volvacea was ascertained with the help of malachite green-resistant mutants and aspartate requiring auxotrophs (Chang et al. 1991). In this mushroom the heterokaryotic mycelium can be raised from a single spore containing a single post-meiotic nucleus. However, the primary homothallism assigned to Volvariella spp. is still not final because of variability in single spore isolates. The secondary homothallism is similar to that of unifactorial heterothallic species, but the two spored basidia conceals the possibility of heterothallism and has forced mycologists to designate this typical type of phenomenon as secondary homothallism. The best example of secondary homothallism is found in Agaricus bisporus . The homokaryotic phase peculiar for primary homothallism is found only in spores of three and four spores per basidium and the frequency of occurrence is approximately 5%.

The process of mushroom breeding has remained integral part of all research programs but initially, the process of strain improvement centered round identification and selection of better performing strains. However, with the release of two hybrids U1 & U3 by the conventional techniques of crossing homokaryons and selection of high yielding hybrids (Fritsche 1991), the mushroom breeding programmes all over the world took a big leap and newer techniques supporting the breeding programmes were brought to light (Loftus 1995). The newer techniques, like protoplasting, have also improved the methodology as well as frequency of homokaryons isolation, which have utility in hybrid formation. The distinct features of different mushroom species like existence or non-existence of clamp connections in heterokaryons, variations in number of nuclei per cell and also in basidiospore per basidium have created confusion and forced to adopt different breeding strategies for each species.

In traditional hybridization system of mushroom improvement, the homokaryons obtained either from single spore isolates or from protoplasting, are crossed to make hybrids which are then evaluated for the desirable traits. Though the technique seems to be very simple but, in the absence of variability in the available germplasm, and the dearth of genetic markers to differentiate between the homokaryons and heterokaryons, success has been seldom achieved in obtaining the desired hybrids. But the new developments in the culture preservation techniques and emergence of DNA-based genetic markers have solved many of these problems. The best example of hybridization is the development of Ui and U3 hybrids of A. bisporus (Fritsche 1991) in Netherlands. Similarly, some of the single spore isolates from A. bisporus (Bhandal and Mehta 1989) and Volvariella spp. (Kalra and Phutela 1991) were also reported to have higher yield potential than their parents.

The Agaricus Recovery Programme (ARP) was founded by Dr. Kerrigan in 1988 which has been recently renamed as Agaricus Resource Programme (Kerrigan 2004). Presently, more than 206 Agaricus strains including the wild ones are available with the ARP and have been identified by using isozyme marker techniques. The strains available with ARP are an excellent source of genetic variability and best example is the four spore per basidial strain obtained from the Sonaran desert of California by Callac et al. (1993). Molecular techniques can be helpful in removing the existing species-specific barriers in developing new hybrids of mushrooms. The use of genetic makers is straightforward for monogenic traits that segregate in distinct phenotypes such as cap color in mushrooms (Kerrigan 2004).

Species-specific pairs of primers were also selected for the other Tuber species which, although economically less important, are morphologically similar to the prized white truffle (T. magnatum ) and to the so-called “bianchetto” (or T. borchii ), and are often used in perpetrating commercial frauds. The T. maculatum-specific primers (TmacI and TmacII) gave a specific amplification product of 407 bp present only in the T. maculatum samples. The specific Tmac primers were also tested on ectomycorrhizae obtained from roots of Ostrya carpinifolia Scop. inoculated with T. maculatum (Amicucci et al. 1998). The amplification of the specific region demonstrated that the ectomycorrhizae analyses belonged to the species T. maculatum and confirmed the success of the mycorrhizal inoculation. For this reason, a reliable identification can be performed even when starting from very little DNA (e.g., even from a single root tip), sampled during any phase of the truffle life cycle. The strategy adopted was particularly useful in discriminating among closely related Tuber species of different economic importance. In particular, it provides a fast and simple tool able to distinguish T. magnatum , the most valuable white truffle , from T. borchii , which is edible but less prized, and is morphologically very similar. The pairs of primers selected in this study are of interest for many biotechnological applications (Amicucci et al. 1998) in particular, they can be used to characterize fruit bodies of different truffles.

4 Researches on Mushroom Cultivation

To meet the increasing demands in the different parts of the world mycologists have suggested different cultivation methods. Mushroom cultivation is also promoted among farmers and tribes to provide healthy nutrition and additional income. Grimm and Wosten (2018) advocated use of spent mass substrate (SMS) in different ways in circular economy. Usually each 1 kg fresh mushroom may produce 2 kg dry waste (Finney et al. 2009), which can be used to produce compost (Uzun 2004), feed for animals (Nasehi et al. 2017), extract enzymes and biofuel (Phan and Sabaratnam 2012), produce materials (Jones et al. 2017) and in bioremediation. Thus mushroom production has great potential. It can solve the problem of stubble burning but such industries should not be clubbed in a small zone, which may lead to production of greenhouse gases (Leiva et al. 2015). Popular methods for cultivation include:

• (a) Laboratory cultivation

• (b) Backyard or small-scale cultivation

• (c) Industrial cultivation and

• (d) Intercropping method in agriculture fields.

Jaykumar and Manoharan (2014) from Coimbatore, India suggested inter-cultivation of paddy straw mushroom in maize cropping system. Maize and cowpea were grown in a field, row spacing with 60 cm row spacing. After 45 days cowpea was cut and bundles of moist paddy straw were placed and crop humidity was 65–68%. Horse gram (Dolichos biflorus) powder was sprayed and inoculation with Volveriella volvacea spawn was done. Water was sprinkled daily and after 10 days fruit bodies were harvested with Biological Efficiency (BE) of 5% (Fig. 16.5).

Fig. 16.5

B.E. of six different mushrooms at different temperatures

4.1 Lenzites sterioides: Structure and Cultivation

The sporophores are sessile, attached by board base, usually imbricate, corky, 8 × 12 cm, 0.3–0.8 mm board upper surface brownish red, zonate, context cinnamon, corky (Fig. 16.6a). The 1–4 mm thick, hymenial surface, gray, pores usually lamellar (Fig. 16.6b). Lamellae are regular, close, basidia -clavate, basidiospores hyaline, cylindric, apoculate 7–11 × 3–3.7μm, hyphal body dimictic. Lenzites spp. are wood-decay , white-rot fungi (class Agaricomycetes, order Polyporale, family Polyporaceae, and genus Lenzites). Lenzites spp. Along with lentinan and schizophyllan, antitumor glucans from G. frondosa and Trametes versicolor have been used in different clinical trials and led to an increase of survival rate and time in patients of different types of cancer, when used in combination with chemotherapy and/or surgery (Lindequist et al. 2005). These wood rotting Lenzites spp. were circumscribed by Elias Magnus Fries in 1835 and were found in parts of Europe, Asia, and Africa. The species produces corky fruiting bodies in the shape of semicircular plates formed on the trunks of several types of deciduous trees. The fruiting body has a lamellar fruit layer (gills) producing spores. The upper surface of the cap may be in various shades of brown and sometimes zonate. The pore surface is white to tan at initial stage but as the fruit body matures, some of the pore walls break down, forming slits with blunt partitions. This results in the characteristic of a mazelike (daedaloid or labyrinthinine/labyrinthiform) appearance. The tube walls are 10–30 mm long with thick walls. Lenzites spp. are widely available and Index Fungorum has reported 26 species.

Fig. 16.6

Basidiocarp of Lenzites. (a) Dorsal view. (b) Lower surface of context showing tubes in gill like manner

4.1.1 Studies on Growth of Three Different Fungi

• (a) Isolation and Procurement of Fungal Cultures

  Isolation was done from fruiting bodies collected from Arboretum of the Botanical Garden, The M. S. University of Baroda, Vadodara. The culture of Lenzites steroids was purified and maintained on PDA slants. The pure cultures of Lentinus edodes and Trametes versicolor were obtained from NRCM, Solan (H.P.).

• (b) Selection of Suitable Culture Medium

  In order to find out the best suitable medium for the growth of three different mushrooms, eight different medicinal nutritional media were tried. The radial colony diameter method was used to record growth. It is evident from Table 16.2 that no growth was recorded in Lenzites and Trametes in Czapek’s medium. Maximum colony diameter of Lenzites was obtained in Malt extract agar followed by Yeast malt and maize extract media. For Trametes and Lentinus sorghum extract was found to be the most suitable medium.

• (c) Selection of Suitable Substrate for Cultivation

  Seven different combination of substrate were tried to find out the best substrate for the cultivation of three mushrooms. Saw dust mixed with 75% Parthenium failed to produce any growth. In substrate mixed with Calotropis dry leaves slow colonization was observed. Table 16.3 shows that the better colonization of Lenzites was recorded in saw dust mixed with maize cake, followed by sorghum and pearl millet.

• (d) Biological Efficiency of Mushrooms

  Use of saw dust and sorghum resulted in the formation of 9–15 fruiting bodies in first flush and after 10 days, four to eight new fruiting bodies were collected and the biological efficiency was 40%. The biological efficiency of other mushrooms under a range of temperatures is depicted in Fig. 16.4.

  It is evident that the BE of six mushrooms was better between 25 and 35 °C. Trametes could not grow at 10 °C. Other three mushrooms Lentinus, Lenzites, and Trametes failed to grow at 40 °C.

Table 16.2 Measurement of fungal growth (cm) in different media

Table 16.3 Substrate selection for cultivation of Lenzites

4.2 Trametes versicolor : Structure and Cultivation

A member of Aphyllophorales the sporopores of Trametes versicolor are sessile, refluxed or umbilicate, sometimes, substipate, corky usually 5–6 × 3.5 × 0.5 cm. upper surface is white strongly concentrically zonate. Context white when fresh ochraceous on drying, soft, 3–5 mm. pore regular , circular to slightly angular, pore tube white, basidia are clavate, 4–5μm broad, basidiospores hyaline subglobose to short ellipsoid, 3–3.5μm.

For lab cultivation of Trametes versicolor saw dust, maize flour and sorghum were used. The substrate was filled in bags and was incubated at 25 °C. Fruiting body were observed after 3 months. The formation of 10–14 fruiting bodies was observed in 2 kg dry saw dust. The biological efficiency was 20–30%. The BE of mushrooms was better at 30 and 35 °C but no fruiting occurred at 10 and 40 °C. Detailed results are depicted in Fig. 16.5.

4.3 Lentinula edodes : Structure and Cultivation

Shiitake mushroom, the common Japanese name for Lentinula edodes (Fig. 16.10d), derives from the mushroom associated with the shii tree (Castanopsis cuspidate Schottky) and take, the Japanese word for mushroom (Table 16.1). Because Japan is the world leader in production of this type of mushroom, the mushroom is now widely known by this name. This mushroom has been renowned in Far East countries (e.g., Japan, China, Korea) as a food and medicine for thousands of years. In the year 199 A.D., Kyusuyu, a native tribe of Japan, offered the Japanese Emperor Chuai a shiitake mushroom. Even older documents record its use in ancient China, where it was referred to as “ko-ko” or “hoang-mo” (Hobbs 1995). The cultivation of this mushroom has been practiced for a thousand years, with its cultivation originating in China during the Sung Dynasty (960–1127). Both history and legend credit Wu San Kwung as the originator of shiitake cultivation. Almost every mushroom-growing village in China has a temple in his honor (Miles and Chang 1997). In 1313, Chinese author Wang Cheng recorded shiitake-growing techniques in his Book of Agriculture. He described how to select a suitable site, choose appropriate tools, and cut down the trees on which one could cultivate the mushrooms. He outlined the basic methods as follows: Cut the bark with a hatchet and cover the logs with soil. After 1 year, top the soil and water frequently. Beat the logs with a wooden club to induce mushroom production. The mushrooms will appear after a rain (Chang and Miles 1992). The Lentinula was introduced to Japanese farmers by the Chinese between 1500 and 1600 A.D. (Chang 1999).

4.3.1 Cultivation of Lentinula edodes

Arya and Arya (2003) reported synthetic log method of shiitake cultivation. Saw dust (80%) consisted of forest trees like Tectona grandis , Acacia arabica, and Terminalia catappa, 10% each of pearl millet and wooden wool, 1 g/kg brewer’s yeast and 100 mg Bavistin. The pH was adjusted to 6.0. The substrate was filled in polypropylene bags (30 × 45 cm), autoclaved and incubated at 20 °C in darkness. The saw dust was completely covered with mycelium in 120 days. Warty out growths appeared on 140 days (Fig.16.10a). The young buttons were reddish brown in color after 170 days of spawning these young buttons opened to form umbrella like thick white pileus (Fig. 16.10b). The C:N ratio was 300:1 of the substrate which changed to 190:1 after 140 days. The pH changed from 6 to 5.5 after 160 days. In first flush, 30 mushroom bodies were formed. Good yield of 45% Biological efficiency was obtained at 15 °C. Light was found necessary for initiating fruiting. Royse and Sanchez-vazquez (2000) observed influence of wood chip particle on production of mushrooms in synthetic logs.

According to Chen (2001) lower the CO₂ in the bag, when bumps become too numerous by cutting slits on the bag. In any case, some aeration should be provided when bumps are formed. Browning of Pileus: There are two different approaches, browning outside of the bag vs browning inside of the bag. Some growers remove the entire bag when browning covers 1/3–1/2 of the mycelial coat in the bag (Oei 1996). Royse (1997) recommended removing the bags before pigmentation, thus allowing browning to occur outside of the bag. Timing of bag removal is crucial. Yield can be affected if bag removal is too early or too late. Maintain 60–70% R.H. to avoid contamination after bag removal. Air enhances browning. Mycelia turn reddish brown at the surface, when exposed to air and eventually forms a dark brown protective, dry, and hardened surface which functions like a tree bark. The inner substrate becomes soft and moist as a consequence of fungal metabolism. Cultivation bags with supplemented substrate (top); colonized substrate blocks with whitish mycelia in cultivation bags (bottom). Browning (pigment formation) initiation and completion in bags is tried. At that time moisture content can be as high as 80% (Oei 1996), ideal for fruiting formation. Apply fruiting induction when spawn reaches physiological maturity and after browning and bark formation. Water soaking is commonly used for fruiting induction after browning and bark forming. On expanding (opening) of the mushroom cap, color becomes lighter.

4.4 Ganoderma lucidum : Structure and Cultivation

Reishi mushroom is a hard woody fungus with red laccate upper surface of pileus. The bodies are raised in black stalk and the basidiocarps are large with truncate basidiospores. Ganodermic acid and glycoproteins with mutagenic and immunomodulatory activity are isolated from fruiting bodies and mycelium. In China and Korea, G. lucidum known as Ling Zhi which means "herb of spiritual potency" whereas the Japanese call this mushroom Reishi or mannentake (10,000 year mushroom) (Chang 1993). In Latin, lucidum means shiny or brilliant and aptly describes this mushroom’s fruiting body, which has a modeled, sculptured, varnished appearance. The virtues of G. lucidum extracts, handed down from generation to generation, include it as a “cancer cure” and a symbol of happy augury, good fortune, good health, longevity and even immortality (Hobbs 1995). Over the years, at least 166 laccate Ganoderma species have been described worldwide, of which at least 48 names were, at some point, considered to be synonyms of others. Based on molecular phylogenetic evidence, it appears that most collections labeled G. lucidum in North America do in fact best correspond to the taxon labeled G. resinaceum in Europe, whereas G. tsugae in North America is genetically very close to the “true” G. lucidum from Europe (Moncalvo 2000). Yao et al. (2020) suggested that, G. lucidum , has been misapplied to this species for over 100 years until recently identified as G. sichuanense. Soon after this, a new species name, G. lingzhi , was also proposed for the fungus because of an unusual internal transcribed spacer (ITS) sequence purportedly of the holotype of G. sichuanense.

• (a) Effect of culture media on the growth of Ganoderma

  • 1. Most mycologists develop preferences for certain types of media based on experience and peculiarities of the type of fungi they have grown. Media affects colony morphology, color, and stage of fungal culture to grow. Different fungi lack the necessary enzymes to utilize different carbon sources. All fungi may require specific elements for growth and their reproduction. Effect of two culture media PDA and PMA was observed on the growth of two strains GN and G29 of Ganoderma lucidum . The growth was more in G29 on PDA and of GN strain on PMA detailed results are shown in Fig. 16.5.

• (b) Following broth media were tried

  • 1. GMY = Glucose 20 g. Malt Extract 20 g. Yeast Extract 1 g

  • 2. AMY antibiotic = (Chloramphenicol) Malt Extract 20 g. Yeast Extract 1 g.

  • 3. STMY = Starch, 20 g. Malt Extract 20 g. Yeast Extract 1 g

  • 4. SMY = Sucrose 20 g. Malt Extract 20 g. Yeast Extract 1 g

  • 5. MMY = Mannose 20 g. Malt Extract 20 g. Yeast Extract 1 g

  • 6. MY = Malt Extract 20 g. Yeast Extract 1 g

  Different carbohydrates are essential to provide energy and are used by fungi with different rates. Basidiomycetous fungi prefer maltose than glucose. Detailed results are presented in Table 16.4 and Fig. 16.5. It is evident from the above table that strain GN grew better in STMY medium followed by MMY and AMY while strain G29 grew better in medium MMY followed by STMY and AMY (Fig. 16.7).

• (c) Laboratory Cultivation Trials of Ganoderma

  Cultivation trials were conducted by Arya (2007) on two strains of G. lucidum one from natural isolation and other procured from NRCM Shimla (Fig. 16.8). Sawdust of trees like Tectona grandis , Acacia arabica and Terminalia catappa was mixed with 10% corn meal and 10% pearl millet. Moisture content was 65% the bags were sterilized, inoculated and kept in dark at 30 °C (Fig. 16.9a). The pH of substrate was 5.5. After complete colonization the bags were shifted in to the culture room. The room has 95% humidity, 1000–12,000 ppm CO₂ and light of 500 lux. On fluffy mycelium warty growth appeared after 68–70 days of inoculation and after 6 days of opening the bags (Fig. 16.9b) After 80 days, young buttons turned red. At that time moisture level was reduced and pileus got flattened and margins of basidiocarp were white initially. The mature fruiting bodies turned red in 90 days. Complete cycle to from the bodies was 90 days. Biological efficiency was 15–20%. The strain which was procured from nature failed to produce any fruiting bodies in 90 days (Fig. 16.10).

Table 16.4 Increase in fungal biomass of two strains of Ganoderma in six different media

Fig. 16.7

Histogram showing growth (mg) of G. lucidum after 15 days on six different culture media. (Source: Arya et al. 2020 Env. at Crossroads p. 7)

Fig. 16.8

Mushrooms of Ganoderma . (a) Ganoderma showing laccate surface. (b), (d) and (e) Different shapes of basidiocarp attached to tree. (c) Tube layer in section and basidiospores

Fig. 16.9

Cultivation of Ganoderma . (a) Bags filled with saw dust and wood fillings. (b) and (c) Production of bud like structures. (d) Expansion of basidiocarp. (Source: Arya 2007)

Fig. 16.10

Cultivation of Lentinus. (a) Pin head formation. (b) and (c) Growth of fruiting body. (d) Mature fruiting bodies of Lentinus edodes

5 Remediation Studies

Biosorption varies with metals. This mushroom is a potential source of raw material for the development of biosorbent. Dry fruiting bodies of Ganoderma lucidum , which are medicinally important were used for bioremediation. A part of the fruiting body was crushed in a grinder. The 2 g of the powdered mass of Ganoderma lucidum was mixed with 100 ml of 100, 500 and 1000 ppm of Cd salt. The solution was prepared from the cadmium sulfate (extrapure) salt (3CdSO₄ · 8H₂O) in 100 ml of distilled water in three different concentrations. Wild mushrooms, grow wild in all types of environments ranging from forests to polluted soils and water bodies. With biosorption they uptake the metals in their fruiting bodies, mycelia and sporocarps. Biosorption of various metals by different mushrooms like chromium by G. lucidum (Das et al. 2008), copper by Lentinus and Lenites (Muraleedharan et al. 1995) and cadmium, mercury and copper by Agaricus macrosporus (García et al. 2005) has been investigated.

In order to use white-rot fungi successfully for bioremediation, knowledge must be utilized from fungal physiology, biochemistry, enzymology, ecology, genetics, molecular biology, and engineering, among other cognate subjects. A four-phase strategy has been advocated (Lamar and White 2001): bench-scale treatability, on-site pilot testing, production of inoculum, and finally full-scale application. For inoculum production substrates like wood chips, wheat straw, peat, corn cobs, sawdust, a nutrient-fortified mixture of grain and, bark, rice, annual plant stems and wood, fish oil, alfalfa, spent mushroom compost, sugarcane bagasse, coffee pulp, sugar beet pulp, okra, canola meal, cyclodextrins, and surfactants can be used (Singh 2006). It is critical to attain the correct nitrogen/carbon ratio in the substrates used, so as to avoid any impeding effect on the efficiency of the fungi in the bioremediation process. The latter approach, termed encapsulation, is derived from the mushroom spawn industry, and both preserves the viability of the inoculum and contributes nutrients to maximally support the degradation of pollutants. Fungal inocula coated with alginate, gelatin, agarose, carrageenan, chitosan, etc., in the form of pellets, may offer a better outcome using bulk substrates. This increases the survival and effectiveness of the introduced species. Fungal inoculum may also be obtained by solid state fermentation. Such inoculum preparation methods improve the likelihood of success in the first phase (above), while good technical and engineering vitalize the second phase. Success in stages three and four depends on the exact remediation application practices employed for the monitoring, optimization, continuity and maintenance of the process. There are some patents available which refer to the subject of remediation using white-rot fungi (Singh 2006).

The mixture of Ganoderma lucidum and CdSO₄ solution was mixed and placed in Rotary shaker for 20, 40, 60 and 120 min. and after that the solution was filtered using Whatman filter paper no. 1. After filtration the biomass was kept in the oven for drying at 72 °C for 24 h. The dried mass was analyzed by the instrument RDX-500 XRF X-ray fluorescence spectrometer for reduction in concentration of CdSO₄ (Arya et al. 2020). The fungal cell wall exhibits excellent metal biding properties due to its components (Das et al. 2008). It is composed mainly of chitins, mannans, glucans, in addition to lipids, polysaccharides, and pigments e.g., melanin (Abbas et al. 2014). Fungal cell wall is reported to be made up of 90% polysaccharides. The functional groups which are involved in metal binding include carboxyl, phosphate, uranic acids, proteins, nitrogen containing ligands, chitin or chitosan (Huang and Huang 1986). The white-rot fungus Phanerochaete chrysosporium is an ideal model for bioremediation by fungi, since it is more efficient than other fungi or microorganisms in degrading toxic or insoluble materials. It presents simultaneous oxidative and reductive mechanisms which permit its use in many different situations, regarding the type of contamination, its degree, and the nature of the site itself. A number of other white-rot fungi also can degrade persistent xenobiotic compounds, e.g., Pleurotus ostreatus , Trametes versicolor , Bjerkandera adusta , Lentinula edodes , Irpex lacteus , Agaricus bisporus , Pleurotus tuber-regium, Pleurotus pulmonarius (Singh 2006; Adenipekun and Lawal 2012).

The experimental results revealed that reduction was fast in 100 ppm conc. at 40 s. although further reduction in conc. of heavy metal salt was observed at 50 and 100 ppm conc. Similar results have been observed by Suseem and Saral (2014), who studied the efficacy of Pleurotus eous , which has analgesic, anti-inflammatory, and antipyretic properties, in biosorption of heavy metals like Lead, Chromium and Nickel. The experiment conducted on mushroom bodies obtained from Kerala Agriculture University, Trivandrum resulted in pH 5, 3, and 7 for the removal of Pb, Cr, and Ni respectively. The percentage removal of all the heavy metals was found to be biosorbent dependent and found to increase with increase in biosorbent dosage. Terragon oyster mushroom P. eous exhibited maximum uptake of Lead (93.2%) as compared to Chromium (27.6%) and Nickel (38.3%) (Suseem and Saral 2014). An experiment was performed to grow Pleurotus sajor caju in modified Asthana and Hawker’s medium ‘A’ (d-glucose = 10 g/l) 1, 5, 10 ppm of ZnSO₄, CuSO₄ and MnSO₄ were added. Flasks were inoculated and kept at 25 °C for 15 days. The results showed that total biomass produced by CuSO₄ was more at 10 ppm. After 5 days maximum growth was observed at 10 ppm while it was more in case of 1 ppm after 10 days. At high levels of metal concentration, cell membrane can damage, enzyme specificity may change, cellular functions are disturbed and DNA structure gets damaged (Bruins et al. 2000). The analyses performed using inductively coupled plasma atomic emission spectrophotometer showed that in Boletus relative abundant contents of P, K, Fe, Mg, Ca, were detected and Na and less of Zn, Cu, and Mn were present. Caps compared to stalks were enriched in P, K, Cu, Mg, and Zn, while stalks were enriched in Mn (Wang et al. 2015).

Immobilized A. bitorquis had higher heavy metal ions remediation potential as compared to other two strains. The maximum potential of live immobilized A. bitorquis for remediation of Cr(III), Pb (II), Cr (VI), and Cu (II) was 226.6, 208.5, 207.3, and 205.1 mg/g, respectively (Hanif and Bhatti 2013). Reduction of Cd was reported by ectomycorrhizal fungi like Laccaria laccata and Suillus luteus associated with Pinus sylvestris in Belgium (Colpaert et al. 2011).

6 Conclusion

Mushrooms have great potential in many Asian and developing countries of Africa to meet the protein diet requirements in food . A variety of mushrooms are collected and cultivated in India and worldwide. Biology and cultivation methods of four medicinal mushrooms are discussed in detail. Their food , therapeutic and bioremediation potential has been discussed. If the natural resources base is not managed for the long term, and is exploited for short-term gains, it will never help in economic development of different nations. The cultivation methods are helpful in removing the waste generated in agriculture and forestry. Mushroom cultivation is good for ecosystem health and improving financial gains of farmers and youth at rural level. There is a need to develop preservation and processing units to utilize these perishable mushrooms. The mushrooms occurring in polluted atmosphere should not be consumed as they may accumulate carcinogenic heavy metals. However these can be utilized for water purification and soil bioremediation purposes.